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Studies on free radical scavenging ability for the crude extracts of Pogostemon cablin Benth leaves
Get PDF目錄 ... I
圖目錄 ... IV
表目錄 ... V
附圖表目錄 ... VI
中文摘要 ... VII
英文摘要 ... IX
縮寫表 ... XI
第一章 前言 ... 1
1. 自由基 ... 2
2. 抗氧化劑 ... 9
3. 中草藥的應用與開發 ... 16
4. 廣藿香的背景與相關研究 ... 21
第二章 研究方法 ... 27
1. 研究目的 ... 27
2. 實驗材料與設備 ... 28
3. 實驗流程圖 ... 30
4. 實驗方法 ... 32
4.1 廣藿香之水及乙醇萃取物的製備 ... 32
4.2 DPPH自由基清除能力測定 ... 32
4.3 ABTS自由基清除能力測定 ... 34
4.4 廣藿香之蛋白質萃取 ... 36
4.5 硫酸銨沉澱 ... 37
4.6 Acetone沉澱 ... 39
4.7 超微薄膜過濾技術 ... 39
4.8 Lowry蛋白質定量 ... 40
4.9 SDS-PAGE蛋白質電泳 ... 42
4.10 膠片乾燥法 ... 46
第三章 結果 ... 47
1. 廣藿香之水及乙醇萃取製備 ... 48
2. 廣藿香之水及乙醇萃取物之抗氧化能力 ... 48
2.1 DPPH測定 ... 48
2.2 ABTS測定 ... 48
3. 廣藿香水及乙醇萃取物經加熱及酸鹼處理之抗氧化能力穩定度 ... 49
4. 廣藿香蛋白質萃取純化 ... 50
5. 蛋白質定量 ... 50
6. SDS - PAGE蛋白質電泳 ... 51
7. 廣藿香蛋白萃取之自由基清除能力測定 ... 51
8. 蛋白質鑑定 ... 51
第四章 討論 ... 53
第五章 結論 ... 57
參考文獻
圖目錄
圖一 、 BHA之DPPH自由基清除能力標準曲線 ... 59
圖二 、水萃取及乙醇萃取之DPPH自由基清除能力 ... 60
圖三、水萃取及乙醇萃取之ABTS自由基清除能力 ... 61
圖四、廣藿香的水萃取及乙醇萃取經熱及酸鹼處理後在DPPH測定之抗氧化的穩定 ... 62
圖五、廣藿香的水萃取及乙萃取經熱及酸鹼處理後在ABTS測定之抗氧化的穩定度 ... 63
圖六、粗純化之蛋白質定量 ... 64
圖七、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)之蛋白質分析 ... 65
圖八、粗純化蛋白之DPPH及ABTS測定 ... 66
圖九、蛋白質多序列比對結果 ... 67
表目錄
表一、廣藿香水及乙醇萃冷凍乾燥物之萃取率 ... 58
附圖表目錄
附圖一、自由基的形成 ... 3
附圖二、自由基氧化損傷機制圖 ... 5
附圖三、抗氧化系統的機制 ... 10
附圖四、抗氧化劑的分類 ... 11
附圖五、穀胱甘肽還原氧化途徑 ... 14
附圖六、廣藿香不同品種圖 ... 22
附圖七、廣藿香中九個確定的化合物結構 ... 25
附圖八、抗氧化劑與DPPH自由基反應機制 ... 33
附圖九、 ABTS反應機制 ... 35
附圖十、硫酸銨飽和濃度 ... 38
附圖十一、超微薄膜過濾裝置 ... 40
附表一、與氧自由基相關的一些臨床疾病 ... 8[[abstract]]先前發現廣藿香(Pogostemon cablin Benth)皂與迷迭香皂比較下，廣藿香皂其本身顏色(綠色)不易氧化，且對色彩之維持更穩定。發現色彩鮮艷的蔬果上，通常有很好的抗氧化能力，因此推測廣藿香應具有抗氧化力的存在。本研究以廣藿香葉片合併液態氮研磨成粉末，分別以水及95 %乙醇萃取後進行凍乾，水和乙醇提取物分別進行DPPH (1,1-diphenyl-2-picryhydrazyl)和ABTS (2,2-azino-bis(3-ethylbenzthia-zoline-6-sulfonate))進行抗氧化活性測定。結果顯示在DPPH方面水萃取物(1.5 mg/ml)與乙醇萃取物(0.25 mg/ml)的自由基清除率，分別為91 %及92 %；在ABTS方面水萃取物(3 mg/ml)與乙醇萃取物(1.5 mg/ml)的自由基清除率，分別為47 %及96 %。廣藿香粗萃取液經由分別加熱60℃，15分鐘或100℃，15分鐘及酸反應30分鐘處理下，測定DPPH，水萃取變異改變率平均約0.07 %，乙醇萃取則約0.01%；測定ABTS在相同處理下，水及乙醇萃取變異改變率平均分別約0.25 % 及0.05 %。然而， DPPH及ABTS清除自由基活性在鹼的處理下無法計算出清除率。原因是鹼液本身就會消退DPPH 及ABTS的吸光值，由上述得知，經加熱處理的廣藿香萃取物，對其抗氧化能力之損失影響很小，故推測其抗氧化力成分中的蛋白質影響力是不重要的或是其蛋白質的3D 結構不為影響其抗氧化力的重要因子。為了解蛋白質在其抗氧化能力的角色，因此初步進行蛋白質簡單純化及活性測試。廣藿香蛋白質萃取經硫酸銨沉澱追蹤其抗氧化能力，在50-70 % 硫酸銨沉澱片段被測到清除自由基活性，DPPH清除率約70 %；ABTS清除率約50 %，經SDS page預估其蛋白質分子量約為30 kDa，並送至中興大學蛋白質體核心實驗室進行蛋白質質譜分析身分鑑定，得到七個可比對到的胺基酸序列片段，但蛋白質序列比對結果發現這七個片段並非對應到同一個蛋白質，極有可能是個新蛋白或送去身分鑑定的蛋白質純度不足所造成的結果，這可能會是未來需要釐清的地方。
Previously, the P. cablin handmade soap can maintain the color (green) longer than rosemary soap. In general, it was found the antioxidant capacity existed in bright color of vegetable and fruit. Therefore, We speculated the P. cablin might contain the antioxidant capacity. In this study, P.cablin leaves were ground to powder with liquid nitrogen and then extracted their components with water or 95 % ethanol for freeze-drying. The antioxidant activity of the extracts from water and ethanol were determined by DPPH (1,1-diphenyl-2-picryhydrazyl) and ABTS (2,2-azino-bis(3-ethylbenzthia-zoline-6-sulfonate)) method respectively. The results showed that the free radical scavenging capacity of water extract (1.5 mg/ml) and ethanol extract (0.25 mg/ml) in DPPH assay, 91% and 92% respectively. That of water extract (3 mg/ml) and ethanol extract (1.5 mg/ml), 47% and 96% respectively in ABTS assay. The average change rates of scavenging activities were 0.01% from water extraction and 0.07% from ethanol extraction temperature treatment (at 60℃ for 15 min, 100℃ for 15 min), or acid treatment for 30 min in DPPH assay. The rates of average change were also detected by ABTS in scavenging activities and the rates were 0.05% and 0.25% respectively from water and ethanol extraction in the same treatment. However, there were not counted the scavenging activities in alkali condition by DPPH and ABTS methods. The reason was the optical absorption of DPPD or ABTS reaction mixture would decay in alkali treatment. There was little effect in scavenging activities of sample treatment with heat, so the protein effect or the 3D structure of protein might not be important factor for antioxidant activity. To understand the role of protein in free radical scavenging capacity, the fragments that the protein was purified by ammonium sulfate precipitation then monitored its scavenging activity. The protein was detected scavenging activities rate about 70% in DPPH assay and about 50% in ABTS one in the 50-70% ammonium sulfate precipitation, This protein molecular weight estimated 30 kDa approximately by SDS page. The protein was identified with mass spectrometry by National Chung Hsing University proteomic MS core laboratory, there were seven fragments of amino acid sequence were obtained and aligned by NCBI web. The result of amino acid sequence alignment was shown the protein might be a novel protein or a mixed protein. This question need be clarified in future hopefully
Effect of phospholipid hydroperoxide glutathione peroxidase (PHGPx) on cell migration in human epidermoid carcinoma A431 cells
No full text口試委員審定書
致謝--------------------------------------------------------------------ii
中文摘要--------------------------------------------------------------iii
英文摘要---------------------------------------------------------------v
目錄-------------------------------------------------------------------vii
圖目錄-----------------------------------------------------------------ix
縮寫對照表------------------------------------------------------------x
第一章 緒論
前言--------------------------------------------------------------------1
麩胱甘肽過氧化酶(glutathione peroxidase; GPx)--------------2
硒-----------------------------------------------------------------------3
基質金屬蛋白酵素（Matrix metalloproteinases; MMPs)-----3
細胞移行--------------------------------------------------------------6
PHGPx的生理功能-------------------------------------------------9
實驗目的與設計-----------------------------------------------------9
第二章 材料方法
藥品材料--------------------------------------------------------------13
子宮頸上皮癌細胞(A431)之培養--------------------------------16
Cell lysate 製備-----------------------------------------------------16
西方墨點法(Western blotting)------------------------------------18
Wound healing------------------------------------------------------21
Transmigration assay----------------------------------------------22
Glutathione assay--------------------------------------------------23
第三章 實驗結果
PHGPx大量/降低表現之穩定細胞株-----------------------26
PHGPx含量對細胞傷口癒合的影響------------------------26
PHGPx含量對細胞轉移表現的影響------------------------28
PHGPx含量對細胞中MMPs表現量的影響--------------28
PHGPx本身的抗氧化能力對細胞移行之影響------------29
PHGPx可促進細胞移行的能力是否與它本身即是結構蛋白有關-------------------------------------------------------------30
第四章 討論與結論
PHGPx大量/降低表現之穩定型細胞株--------------------32
PHGPx大量表現與降低表現對細胞的影響---------------33
細胞中PHGPx之表現量對細胞運動性的影響------------33
PHGPx之抗氧化能力與協助細胞運動之關係------------34
PHGPx表現量與MMP 9以及MMP 2的關係------------36
PHGPx本身是否也屬於結構蛋白之探討------------------36
參考文獻----------------------------------------------------------38
圖表----------------------------------------------------------------45
附錄----------------------------------------------------------------68[[abstract]]磷脂氫穀胱甘肽氧化酶(PHGPx)本身具有極強的抗氧化作用，目前已知它不僅可保護細胞膜免遭受過氧化的損傷，而且還可能參與細胞訊息傳遞的調控。基質金屬蛋白酵素 9（Matrix metalloproteinase 9: MMP 9）屬於MMPs的家族成員之一，它與腫瘤的轉移特別有關。研究指出幾乎所有的癌細胞皆會有細胞移行的作用。
之前實驗室的研究發現，在多種癌細胞中，PHGPx的mRNA表現皆有較高的趨勢。至於本篇論文，主要在探討癌細胞中大量表現之PHGPx是否與癌細胞的轉移有關，而其機制又是如何？本篇所使用的細胞株有三：分別為vector control穩定型細胞株(TR 15)，inducible PHGPx-overexpression穩定型細胞株(IA 38)，以及inducible PHGPx-knockdown穩定型細胞株(IPI 7)。
經由傷口癒合的實驗發現：當細胞大量表現PHGPx之後的癒合能力明顯比PHGPx 被降低之細胞要來得快。另外，由Western blot的結果顯示，PHGPx 大量表現的細胞其MMP 9的表現量相對比PHGPx knockdown之細胞要來的高，此意味著細胞中的PHGPx大量表現時，所促使細胞移行加速的機制是需要透過MMP 9的表現方能達成。至於MMP 2，由於Western blot的結果顯示其變化量不大，所以可以排除其與PHGPx造成細胞移行加速之關係。
在探討作用機制方面，當細胞額外添加H2O2時，雖然會導致大量表現PHGPx的細胞，其細胞移行的能力會下降，但相較之下，PHGPx knockdown的細胞則下降的更加明顯，由此說明，PHGPx能促進細胞移行的作用是與其具抗氧化能力有關。另外，經由transmigration assay的實驗發現，以diethyl maleate (DEM)去抑制PHGPx酵素反應所需之麩氨基硫後，可觀察到PHGPx大量表現的細胞，其transmigration的能力會比control細胞來的好。而PHGPx knockdown的細胞，則其transmigration的能力會比control細胞更差，由此說明，細胞中的PHGPx在喪失抗氧化活性之下，也是能促進細胞的transmigration能力。由於細胞移行時必須要有結構蛋白的參與方能達成，所以此結果是否暗示了PHGPx可能也屬於細胞內的結構蛋白，因此當其大量表現時即會對細胞移行有正向的幫助。
綜合以上的結果，我們的結論是細胞中的PHGPx具有促進細胞移行之作用，此作用的達成除了與MMP 9的表現有直接相關外，PHGPx本身具抗氧化能力與可能是結構蛋白的細胞功能也是促成的因素之一。
Abstract
Phospholipid hydroperoxide glutathione peroxide (PHGPx) owning powerful antioxidative ability may not only protect cell membrane from the damage of oxidative stress but also regulate the cell signal transduction. MMP 9 belongs to one of the matrix metalloproteinases is interrelated particularly to the tumor metastasis. As indicated in some reports, cell migration may be an important characteristic feature in almost all cancer cells.
In our previous study, it indicated that highly PHGPx mRNA was expressed in a variety of cancer cells. Therefore, the relation and mechanism between PHGPx and tumor metastasis was verified in this study. In this study, we used various inducible PHGPx-expressed transfectants, including overexpression (IA38), knockdown (IPI7) and vector control (TR15).
In wound healing assays, we found that the healing speed in PHGPx-overexpressed cells were significantly quicker than in PHGPx-knockdown cells. In addition, the Western blot results indicated that the MMP 9 expression was higher in PHGPx-overexpressed cells than in PHGPx-knockdown cells. This meant that PHGPx prompting cell migration might be through the expression of MMP 9. As MMP 2, the Western blot results showed less variation, so we excluded the possibility of MMP-2 participating in PHGPx-mediated cell migration.
When supplement with H2O2, the ability of migration was lightly decrease in PHGPx overexpressed cells, however, a significant decrease was observed in PHGPx-knockdown cells. This meant that PHGPx prompting cell migration might also depend on its antioxidant capacity. In transmigration assays, after using diethyl maleate (DEM) to deplete intracellular glutathione that was necessary for the enzyme activity of PHGPx, we found that the transmigration ability of PHGPx-overexpression cells was better than the control cells. Relatively, the transmigration ability of control cells was better than the PHGPx-knockdown cells. These results indicated that PHGPx prompting cell transmigration might not only depend on its antioxidant capacity. As cell migration required the involvement of structure proteins, it might imply that PHGPx belonged to a structure protein and when PHGPx overexpressing would help cell migration.
Based on our results, we concluded that PHGPx promoting cell migration might rely on three factors : Frist, inducing the expression of MMP 9. Second, it has antioxidant capacity. Third, it may be one of the structural proteins
Performance Assessment Of Exterior-Hood Ventilation System With Air Multiplier
No full text第一章 前言
1-1 研究背景
1-2 研究目的
第二章文獻回顧
第三章研究方法
3-1 研究架構
3-2 實驗設備
3-2-1 外裝式氣罩、空氣倍增器與風量量測儀器
3-2-2 定性實驗─流場可視化技術之儀器設備
3-2-3 定量實驗─二氧化碳去除效率評估之儀器設備
3-3 實驗方法
3-3-1 局部排氣系統與空氣倍增器的風量量測
3-3-2 定性實驗─流場可視化技術
3-3-3 定量實驗─二氧化碳去除效率評估
第四章結果與討論
4-1 局部排氣系統與空氣倍增器之風量測試結果
4-2 定性實驗-流場可視化技術測試結果
4-2-1 空氣倍增器之測試結果
4-2-2 外裝式氣罩之測試結果
4-2-3 外裝式氣罩加裝空氣倍增器之測試結果
4-3 定量實驗-二氧化碳去除效率評估結果
4-3-1 外裝式氣罩之評估結果
4-3-2 外裝式氣罩加裝空氣倍增器之評估結果
4-4 討論
第五章結論與建議
5-1 結論
5-2 建議
參考文獻[[abstract]]本研究以流場可視化技術及利用質量平衡方程式，分別利用水霧排除效果與二氧化碳氣體之去除效率進行定性與定量評估，以探討外裝式氣罩加裝空氣倍增器前後的流場變化及對去除效率的影響。本研究利用變頻馬達控制外裝式氣罩加裝空氣倍增器之局部排氣系統在不同的排氣與送氣風量的組合條件下，在氣罩開口面中心線的不同垂直距離處分別設置水霧及二氧化碳釋放源，並固定空氣倍增器與釋放源距離為15公分的情況下，拍攝水霧的排除效果以及控制二氧化碳釋放量條件下，在排氣導管內測定二氧化碳濃度，利用質量平衡方式計算二氧化碳去除效率。由實驗結果得知，未加裝空氣倍增器時，氣罩與釋放源的距離在60公分的垂直距離下，其排除效果不佳，且去除效率已明顯降低；氣罩加裝空氣倍增器於60、90公分的垂直距離下，氣罩的排氣風量大於空氣倍增器的送氣風量時，其排除效果與去除效率均有顯著的提昇，然而當氣罩的排氣風量小於空氣倍增器的風量時，則會造成外裝式氣罩通風系統來不及捕集，而有逸散的情形發生。綜合上述，未來應用空氣倍增器以改善既有外裝式氣罩對於空氣污染物的抽吸排除效率時，控制排氣風量大於送氣風量，在節能與污染物排除將具有顯著助益。
Local exhaust ventilation (LEV) is available engineering control method in air contaminants removal, which with exterior hood was well known for poor capture efficiency. This study applied laser light assisted water-mist flow visualization and used tracer gas decay method by CO2 concentration monitoring with mass-balance principle to evaluate the performance of exterior hood with an air multiplier. In present study, LEV with exterior hood controlled by converter, laser light assisted water-mist flow visualization and CO2 releasing system was applied to monitor CO2 concentrations in emission source and exhausted air. Air contaminants removal performance and efficiency were evaluated by digital-video-camera shoot the case of water-mist removal and mass-balance principle to assess qualitative performance of local exhaustion system with or without air-multiplier under four different distance conditions. The greater of LEV system exhausted air volume induced more efficient of CO2 removal efficiency in a fixed released rate and water-mist removal performance. For LEV exhaust air volume is greater than the aspiration volume from air multiplier, which will increase water-mist removal performance and CO2 removal efficiency. The CO2 removal efficiency and water-mist removal performance are not significant in CO2 source and water-mist source release from one hood-diameter distance without air multiplier. The CO2 removal efficiency and water-mist removal performance are improved significantly with air multiplier from one hood diameter. For exterior hood exhaust is greater than the aspiration of the air multiplier, air multiplier can improve efficacy of exterior hoods
Development and Antioxidative Activity of Ready to Cook Healthy Food-Dried Sweet Potato Cube
No full text目錄----------------------------------------------------------------------------i~viii
圖目錄------------------------------------------------------------------------~iv~vi
表目錄-----------------------------------------------------------------------vii~viii
謝誌-------------------------------------------------------------------------------i
中文摘要---------------------------------------------------------------------viii~ix
英文摘要----------------------------------------------------------------------x~xii
縮寫表----------------------------------------------------------------------------xiii
第一章:可用於煮飯及粥之乾燥甘藷塊之最適條件開發---------------------------------------------------------------------------------------1
第一節、前言----------------------------------------------------------------1~3
第二節、文獻整理----------------------------------------------------------3~9
一、甘藷介紹----------------------------------------------------------3~5
二、甘藷營養成份----------------------------------------------------5~6
三、加工對於甘藷的影響--------------------------------------------6~9
第三節、材料與方法-----------------------------------------------------9~10
一、研究材料-------------------------------------------------------------9
二、研究方法--------------------------------------------------------9~10
第四節、結果與討論---------------------------------------------------11~21
一、藷塊丁之熱風乾燥與外觀----------------------------------11~12
二、甘藷乾燥塊丁的L*a*b*分析--------------------------------12~15
三、甘藷乾燥塊丁的復水率試驗與品評試驗----------------15~21
第五節、結論----------------------------------------------------------------21
第二章、浸泡異抗壞血酸乾燥甘藷之β-carotene含量與抗氧化分析--45
第一節、前言-------------------------------------------------------------45~46
第二節、文獻整理-------------------------------------------------------47~56
一、甘藷的種類--------------------------------------------------------47
二、自由基與抗氧化物質----------------------------------------48~51
三、甘藷所含機能性成份----------------------------------------52~56
第三節、材料與方法----------------------------------------------------56~60
一、研究材料-------------------------------------------------------56~57
二、研究方法-------------------------------------------------------57~60
第四節、結果與討論----------------------------------------------------60~69
第五節、結論-------------------------------------------------------------68~69
參考文獻-----------------------------------------------------------------------83~89
圖目錄：第一章、可用於煮飯及粥之乾燥甘藷塊之最適條件開發
圖1. Hunter L*a*b*值座標軸-----------------------------------------------------23
圖2.TNG 57經三種溫度熱風乾燥圖------------------------------------------24
圖3.TNG 66經三種溫度熱風乾燥圖------------------------------------------24
圖4. 100 ℃熱風乾燥甘藷成品外觀圖---------------------------------------25
圖5. 新鮮甘藷塊丁烹煮後成品圖---------------------------------------------25
圖6. TNG 57 100 ℃熱風乾燥未殺菁組甘藷塊丁以不同水量煮飯之外 觀圖-----------------------------------------------------------------------------26
圖7. TNG 57 100 ℃熱風乾燥未殺菁組甘藷塊丁以不同水量煮粥之外觀圖----------------------------------------------------------------------------26
圖8. TNG 57 100 ℃熱風乾燥殺菁組甘藷塊丁以不同水量煮飯之外觀圖----------------------------------------------------------------------------27
圖9. TNG 57 100 ℃熱風乾燥殺菁組甘藷塊丁以不同水量煮粥之外觀圖---------------------------------------------------------------------------27
圖10. TNG 66 100 ℃熱風乾燥未殺菁組甘藷塊丁以不同水量煮飯之外觀圖------------------------------------------------------------------------28
圖11. TNG 66 100 ℃熱風乾燥未殺菁組甘藷塊丁以不同水量煮粥之外觀圖------------------------------------------------------------------------28
圖12. TNG 66 100 ℃熱風乾燥殺菁組甘藷塊丁以不同水量煮飯之外觀圖---------------------------------------------------------------------------29
圖13.TNG 66 100℃熱風乾燥殺菁組甘藷塊丁以不同水量煮粥之外觀圖---------------------------------------------------------------------------29
圖 14.未殺菁煮粥組及殺菁煮粥組樣品總接受度-------------------------30
圖 15.未殺菁煮飯組及殺菁煮飯組樣品總接受度-------------------------31
圖目錄:第二章、浸泡異抗壞血酸乾燥甘藷之β-carotene含量與抗氧化分析
圖1.β-胡蘿蔔素標準品線性圖------------------------------------------------70
圖2.甘藷萃取液,注射HPLC分析圖-------------------------------------------71
圖3. 經90 ℃熱風乾燥之TNG 66殺菁組甘藷塊丁萃取液之萃取液DPPH清除百分比-----------------------------------------------------------------79
圖4. 經90 ℃熱風乾燥之TNG 66殺菁組甘藷塊丁萃取液之總酚含量----------------------------------------------------------------------------------------80
圖5. 經90 ℃熱風乾燥之TNG 66殺菁組甘藷塊丁萃取液之類黃酮含量-------------------------------------------------------------------------------------81
表目錄：第一章、可用於煮飯及粥之乾燥甘藷塊之最適條件開發
表1民國95年台灣主要甘藷生產縣市統計----------------------------------32
表2歷年來台灣地區甘藷生產統計-------------------------------------------33
表3甘藷可食部份每100g營養成份分析-------------------------------------34
表4不同處理方式之TNG 57 甘藷塊丁L*值-------------------------------35
表5不同處理方式之TNG 57甘藷塊丁a*值--------------------------------36
表6不同處理方式之TNG 57甘藷塊丁b*值--------------------------------37
表7不同處理方式之TNG 66甘藷塊丁L*值--------------------------------38
表8不同處理方式之TNG 66甘藷塊丁 a*值--------------------------------39
表9不同處理方式之TNG 66甘藷塊丁b*值-------------------------------40
表10 100 ℃ 3.5小時乾燥之TNG 57甘藷塊丁之不同烹煮水量復水率------------------------------------------------------------------------------41
表11 100 ℃ 3.5小時乾燥之TNG 66甘藷塊丁之不同烹煮水量復水率------------------------------------------------------------------------------42
表12未殺菁組粥及殺菁組粥差異性分析------------------------------------43
表13未殺菁煮飯組及殺菁煮飯組差異性分析---------------------------44
表目錄：第二章、浸泡異抗壞血酸乾燥甘藷之β-carotene含量與抗氧化分析
表1人體內之抗氧化物輔助因子----------------------------------------------72
表2 TNG 57甘藷塊丁經80 ℃熱風乾燥之 β-carotene 回收------------73
表3 TNG 57甘藷塊丁經90 ℃熱風乾燥之 β-carotene 回收------------74
表4 TNG 57甘藷塊丁經100 ℃熱風乾燥之 β-carotene 回收----------75
表5 TNG 66甘藷塊丁經80 ℃熱風乾燥之 β-carotene 回收------------76
表6 TNG 66甘藷塊丁經90 ℃熱風乾燥之 β-carotene 回收------------77
表7 TNG 66甘藷塊丁經100 ℃熱風乾燥之 β-carotene 回收-----------78[[abstract]]中文摘要
本研究欲開發可直接放入飯或粥中烹煮食用及儲存性高之甘藷塊。首先採用新化地區台農57 (TNG 57)及台農66 (TNG 66)甘藷，探討經不同溫度、時間熱風乾燥甘薯塊之L*a*b*值。分別經80 ℃(5.5 h)、90 ℃(4.5 h)及100 ℃(3.5 h)熱風乾燥甘藷塊丁之L*a*b*值，各組均以殺菁組之a*與b*值較未殺菁組高，顯示殺菁處理可減少甘藷內色素之破壞，而且以溫度愈高且短時間處理組之a*及b*值較高並接近生鮮甘藷之顏色，但超過100 ℃後會有焦化反應反而表現過深顏色。兩品種則以TNG 66有較佳之外觀顏色。將各組乾燥樣品實際加入飯及粥中烹煮，結果殺菁組成品之顏色均較鮮艷，用於煮飯時TNG 66與TNG 57復水率為40-52 %，且兩品種以殺菁組之復水率較高；用於煮粥時則有更好的復水率約為60-65 %，同樣以殺菁組有較佳之復水率。各組經品評之口感與生鮮組均接近，但復水率越高者，其口感及接受度愈佳且復水後之外觀愈接近生鮮組。綜上所述，以TNG 66品種採用 100 ℃ 3.5 h熱風乾燥並經殺菁處理組為本實驗之最適條件。
機能性研究採用新化地區產出之TNG 57及TNG 66甘藷經80 ℃ 5.5 hr、90 ℃4.5 h、100 ℃3.5 h熱風乾燥處理後分析其β-carotene含量。結果TNG 57組甘藷有接近白色之外觀並均僅含微量β-carotene;而TNG 66殺菁組之甘藷含較高之β-carotene，並以90 ℃ 4.5 h 處理組達到225.2 mg/g (dry weight)最高，未殺菁組則較低。接著以異抗壞血酸(Ascorbic acid)浸泡甘藷塊丁，再以90 ℃ 4.5 h熱風乾燥，結果不論殺菁與否Ascorbic acid均有助於保留兩品種甘藷較多的β-carotene含量，5 % Ascorbic acid浸泡後TNG 57 90 ℃殺菁組之β-carotene由32.4 mg/g提升至44.9 mg/g，而TNG 66組更由225.2 mg/g升至280.7 mg/g。TNG 66甘藷90 ℃熱風乾燥殺菁組對DPPH自由基之清除作用則隨浸泡Ascorbic acid濃度越高而升高，浸泡0、3及5 %之10 ul甘藷萃取液(相當於 0.5 g乾重甘藷)之清除作用約為74.0±1.5、78.2±2.4及84.0±0.4 %；而浸泡5 %之25 ul甘藷萃取液之清除作用則達100 %。甘藷萃取物之總酚及類黃酮含量均隨著抗壞血酸浸泡濃度增加而遞減，推論浸泡Ascorbic acid主要可能是藉由保留β-carotene而表現出抗氧化性。
關鍵詞：色差、甘藷、復水率、殺菁、異抗壞血酸、β-胡蘿蔔素、抗氧化性。
Abstract
This study was to development of sweet potato coube for convenient and can be stored directly into the widely acknowledged by experts in food and nutrition, cooked in rice or porridge，First，sweet potato get from Xinhuain in taiwan area of Tainung 57 (TNG 57) and Tainung 66(TNG 66) varietysweet potato，explore the different temperature, time, hot air drying of sweet potato pieces of L*a*b* values. Were treated with 80 ℃ (5.5 h), 90 ℃ (4.5 h) and 100 ℃ (3.5 h) air drying of sweet potato cube for development L*a* b* values，each group blanching group a* and b* values is higher than the no- blanching group，result show blanching can reduce the damage of sweet potato in pigment，and the higher temperature short-time treatment of a* and b* values higher and close to the color of fresh sweet potatoes, but more than 100 ℃, but the performance of the reaction will be over caramel deep color，between both varieties TNG 66 have better color appearance，dried samples of each group will actually add cooked rice and porridge, the results blanching bright than the color composition of products, when used for cooking and TNG 57 TNG 66 was 40-52 %，and blanching group have higher rehydration rate; for porridge is a better rehydration rate of about 60-65 %，use the same blanching group had better rehydration rates，after sensory evaluation，each group and the fresh group were close, but the higher rehydration rate r, its taste and acceptability of the better and after rehydration closer fresh group. In summary, the TNG 66 species with 100 ℃ 3.5 h hot air drying and blanching treatments is the optimal experimental conditions.
In functional study used the Xinhua of TNG 57 and TNG 66 Sweet Potato by 80 ℃ 5.5 h, 90 ℃ 4.5 h，100 ℃ 3.5 h after hot air drying development of the β-carotene content，Results TNG 57 fleshed showed a nearly white appearance of sweet potato cube and only trace β-carotene content，and TNG 66 sweet potato blanching group have higher β-carotene，and 90 ℃ 4.5 h treatment group β-carotene content for 225.2 mg / g ( dry weight) is the highest, no-blanching group is lower，with different Ascorbic acid Immersion of sweet cube， then 90 ℃ 4.5 h hot air drying, result showed，either blanching or no-blanching Ascorbic acid solution will help the two varieties of sweet potato keep more β-carotene content，5 % Ascorbic acid immersion TNG 57 90 ℃ blanching group of β-carotene content by the 32.4 mg / g increased to 44.9 mg / g，and TNG 66 blanching group is the more content by the 225.2 mg / g increased to 280.7 mg / g，TNG 66 90 ℃ hot air drying of sweet potato blanching group of DPPH radical scavenging is higher with increased of Ascorbic acid immersion concentrations，immersion 0,3 and 5 % Ascorbic acid solution ，10 μl of sweet potato extract (equivalent to 0.5 g dry weight of sweet potato) in scavenging around 74.0 ± 1.5,78.2 ± 2.4 and 84.0 ± 0.47 %; and immersion for 5 % of the 25 μl extract of sweet potato The scavenging effect was as high as 100 %，Total phenol and flavonoids content is decreased with sweet potato immersion of Ascorbic acid concentrations increased，Inference may be soaked Ascorbic acid mainly by preserving β-carotene and antioxidant activity shown.
Keywords: color, sweet potato, rehydration rate, blanching, ascorbic acid, β-carotene, antioxidative activit
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Effects of Soil Organic Matter on Biodegradation of Diesel Oil-Contaminated Soil
No full text摘要................................................... II
Abstract............................................... IV
致謝.................................................... VI
表目錄.................................................. XII
圖目錄.................................................. XIV
第一章 前言.............................................. 1
第二章 文獻回顧.......................................... 4
2.1 國內加油站及大型儲槽調查之背景分析.................. 4
2.1.1 國內加油站數量及大型儲槽數量........................ 4
2.1.2 加油站及大型儲槽可能洩漏源分析...................... 4
2.2 石油碳氫化合物分析................................ 6
2.3 土壤有機質....................................... 10
2.4 柴油的生物復育................................... 14
第三章 實驗材料與方法..................................... 17
3.1 批次一: 以噴灑法造成柴油污染....................... 17
3.1.1 土壤背景調查.......................................17
3.1.2 實驗設計......................................... 19
3.1.3 植種微生物....................................... 21
3.2 批次二:以毒性特性溶出程序（TCLP）造成柴油污染....... 24
3.2.1 土壤背景調查...................................... 24
3.2.2 實驗設計......................................... 25
3.3 柴油降解優質微生物添加............................ 29
3.4 批次操作分析方法................................. 30
3.4.1 土壤含水率 ........................................31
3.4.2 土壤pH值......................................... 31
3.4.3 土壤有機質(SOM)測量............................... 31
3.4.4 柴油(TPHd)分析................................... 33
3.4.5 菌落數分析-平板計數法.............................. 36
第四章 結果與討論......................................... 39
4.1 批次一: 以噴灑法造成柴油污染....................... 39
4.2 批次二:以毒性特性溶出程序（TCLP）造成柴油污染....... 43
第五章 結論與建議........................................ 53
第六章 參考文獻.......................................... 55[[abstract]]本研究目的為探討土壤有機質多寡對微生物降解柴油污染土中總石油碳氫化合物-柴油(TPHd)之影響。目標的污染物為柴油。實驗批次分為兩種批次，批次一的土壤有機質分別為: 2%(KT)、26%(Kpeat26)、41%(Kpeat41) 三種不同有機質，所使用的土壤為油污染復育處理土(KT)與泥炭土(Peat)兩種。批次二有機質分別為2%(Gr2)、5%(Gpeat5)、10%(Gpeat10)、20%(Gpeat20)，所使用的土壤為歸仁土(Gr)與泥炭土(Peat)兩種，然後將處理土經由甲醇滅菌後分成實驗組及控制組，其中實驗組使用生物添加技術添加柴油降解菌株，控制組則不添加菌株。
本研究在方法改良上利用環檢所公告毒性特性溶出程序（Toxicity characteristic leaching procedure，TCLP）的特性，由批次一與批次二試驗發現可以改善土壤與污染物的均質關係。結果方面:批次一共操作了110天結束，批次一初始是以原生菌降解THPd，但得知在第42天添加柴油優勢降解菌後，發現第57天至110天KT批次土去除率從34%提升至89%， Kpeat26批次土去除率從60%提升至91%，Kpeat41批次土從50%提升至93%。由此可知所添加的菌株對於降解TPHd有明顯的貢獻。批次二共操作了212天結束，結果發現TPHd皆於40天前有明顯的降解趨勢，在第65天時結束第一階段降解，從一階反應速率常數k得知，批次土Gr2 k值為0.0340 day-1，Gpeat5為0.0311 day-1，Gpeat10為0.0304 day-1，Gpeat20為0.0249 day-1。由此發現土壤有機質越高其降解速率越慢。總去除率方面Gr2為74%、Gpeat5為73%、Gpeat10為69%、Gpeat20為71%，發現土壤有機質多寡並對影響TPHd並不明顯。由土壤有機質的測定得知，在經過長時間的生物復育下，最初與最終的土壤有機質均無明顯減少的現象，由此結果發現，在生物復育中的微生物並無分解土壤有機質的狀況。從平板計數法發現，土壤中的菌落數會隨著TPHd降解而減少，說明菌落數與TPHd有相關。比較實驗組與控制組TPHd降解情形發現，實驗組第一階段降解結束65天以前，實驗組與控制組降解趨勢有明顯差異，由此可知添加優植菌株對降解土壤中的TPHd有顯著的幫助。未來可以使用分子生物技術來探討高有機質土壤當中降解TPHd的菌落生態。
關鍵字: SOM(土壤有機質)、TPH (總石油碳氫化合物)、生物復育
This paper aims at discussing the influence of the quantity of SOM (soil organic matter) on the biodegradation of TPHd in diesel-polluted soil. The target pollutant is diesel oil, called TPHd thereof, with the initial concentration of 4000 mg/kg. The soil experimented are Gueiren Soil and Peat. Soils are differentiated with the quantity of SOM. They are: Gr2 (SOM 2.2%), Gpeat5 (SOM 4.6%), Gpeat10 (SOM 8.7%), and Gpeat20 (SOM 17.9%). The soil experimented are anticepticized with methyl alcohol and then separated as experimental group and control group. The experimental group is supplied with diesel degrading strain with bioadding technique, while the control group is not supplied. To improve the method applied, this study uses the TCLP( Toxicity characteristic leaching procedure) announced by the Environmental Analysis Laboratory, EPA, to test and refine the homogeneous relationship between the soil and pollutant.
The result shows TCLP’s specialty serves well in refining the homogeneous relationship between the soil and pollutant. This experiment lasts for 212 days. The result shows obvious trend of TPHd degradation for the first 40 days. The first stage degradation ends on the 65th day. We learned from first stage response speed first-order reaction constant k that the k value of Gr 2 soil is 0.0340 day-1,Gpeat5 / 0.0311 day-1, Gpeat10 / 0.0304 day-1, and Gpeat20 / 0.0249 day-1. The higher the concentration of SOM is, the lower the speed of degradation. For the total removal rate, the rate of Gr2 is 74%, Gpeat5 73%, Gpeat10 69%, and Gpeat20 71%. The result show no obvious connection between the amount of SOM and TPHd degradation.
By testing the SOM, we learned that no obvious decrease of the SOM is detected between the initial and final stages after long period of bio-recovery. Therefore, the microorganism in bio-recovery doesn’t degrade SOM. With Plate count, we have found the number of strains decreases with the degradation of TPHd. This phenomenon demonstrates the connection between the number of strains and TPHd. By comparing the TPHd degradations in experimental and control groups, we have found obvious difference between them before the first stage degradation ends on the 65th day. Thus, we conclude that adding superior strains apparently improves the degradation of TPHd in the soil. In the future, molecular biotechnology experiments can help studying the ecology of the TPHd-degrading strains in high SOM soil.
Keywords:
SOM (soil organic matter), TPH (total petroleum hydrocarbons), Bioremediatio
Effect of Cinnamaldehyde on Mitogenesis and Migration in Human Colon Cancer Cells
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第一章、緒論 ················································ 1-16
1、 大腸癌 ··············································· 2
(1)、大腸癌臨床分類與症狀 ····························· 2
(2)、癌細胞生長與擴散 ································· 3
(3)、大腸癌與致癌基因之關聯性 ························· 3
2、細胞週期 ·············································· 4
3、細胞程式性死亡 ········································ 6
3-1、抑制凋亡相關蛋白 ··················································· 7
3-1-1、IAP ···································································· 7
3-1-2、Bcl-2 ·································································· 7
3-2、促進凋亡相關蛋白 ··················································· 8
3-2-1、caspase ······························································· 8
4、大腸癌致病機轉 ········································ 9
4-1、Notch1 調控大腸癌細胞之生長 ··································· 9
4-2、趨化因子 IL-8 與大腸癌細胞之相關性 ·························· 11
4-3、β-catenin 和 IMP-1 與大腸癌細胞增生、遷移之相關性····· 11 5、肉桂醛················································ 15
6、訊息傳導途徑 ································································ 16
7、氧化壓力 ······································································ 18
第二章、材料與方法 ··········································· 19
1、 藥品試劑 ············································· 20
2、 細胞培養與細胞冷凍保存 ······························· 23
3、 細胞計數 ············································· 24
4、 MTT 試驗 ············································ 24
5、 細胞毒性試驗 ································································ 25
6、 製備細胞溶解物 ····························································· 26
7、 硫酸十二酯鈉-聚丙烯醯胺凝膠電泳 ··································· 27
8、 西方墨點法分析 ····························································· 27
9、 DNA 萃取 ···································································· 28
10、瓊脂凝膠電泳分析 ························································ 29
11、傷口癒合定量分析 ························································· 29
12、Caspase 3/CPP32 Colorimetric Assay ····················· 30
13、Cytochrome c Releasing Assay ··························· 31
14、GST Colorimetric Activity Assay ······································· 32
15、生物統計 ····································································· 32
第三章、結果 ················································ 34
第四章、討論 ················································ 41
第五章、參考文獻 ············································· 46
第六章、圖表 ················································ 55[[abstract]]大腸癌導因於大腸直腸中細胞不受控制地增生與擴散。近年來，大腸癌在美國為第二癌症死亡的主要原因。而台灣的老年人口增加、油脂的攝取量增加、纖維的攝取量減少、飲食習慣西化，不但心血管疾病增加，大腸癌患者也持續地增加。肉桂醛(cinnamaldehyde)屬醛類化合物，黃色黏稠狀液體，是由肉桂樹的樹枝樹葉中萃取出的主要且具生物活性的化合物。近期已經有愈來愈多研究指出，肉桂醛不但具有抗菌功效，免疫調節作用，抗真菌活性，亦可緩和糖尿病的腎臟病變。然而許多活體外研究證實經肉桂醛處理後，會對不同的組織細胞產生不同的生物效應，其作用機轉亦十分複雜。
本研究主要目的是探討肉桂醛在人類大腸癌細胞的增生與遷移中所扮演的角色。首先從MTT試驗與細胞計數結果我們發現在HT-29及SW-480兩株人類大腸癌細胞中，若以肉桂醛(100 μM)處理細胞24或48小時，其抑制細胞生長之作用並不明顯。直到肉桂醛(100 μM)處理72小時之後才有明顯地影響，會減少大腸癌細胞30%的存活率。其次，於細胞遷移試驗中我們以肉桂醛(100 μM)、ERK 抑制劑 PD98059 (5 μM)、JNK 抑制劑 SP600125 (2 μM)、
p38 抑制劑 SB203980 (2 μM)、PI3K 抑制劑 LY294002 (5 μM)以及 Akt 抑制劑SH-5(2 μM)處理細胞 72 小時後，發現肉桂醛與這些蛋白激酶抑制劑都會抑制HT-29及SW-480細胞之遷移。有趣的是肉桂醛亦會明顯地削弱ERK/JNK/p38 MAPK 以及 PI3K/Akt 訊息傳遞路徑的活化。因此我們推測肉桂醛會透過抑制上述兩種訊息路徑，進而減少大腸癌細胞的增生與遷移。另一方面，肉桂醛會增加caspase 3活性、抑制Bcl-2 蛋白的表現、促進 PARP蛋白結構的切割、誘發DNA 分子斷裂以及cytochrome c從粒線體被釋放至細胞質，進而導致HT-29及SW-480細胞凋亡。再者，我們更發現肉桂醛會
桂醛會透過抑制上述兩種訊息路徑，進而減少大腸癌細胞的增生與遷移。另一方面，肉桂醛會增加caspase 3 活性、抑制Bcl-2蛋白的表現、促進 PARP
蛋白結構的切割、誘發 DNA 分子斷裂以及 cytochrome c 從粒線體被釋放至細胞質，進而導致 HT-29 及 SW-480 細胞凋亡。再者，我們更發現肉桂醛會導致細胞週期停頓可能是透過減少PCNA蛋白質合成以及增加p27Kip1與p21Waf1/Cip1蛋白質表現所造成的。
因此綜合以上結果發現ERK/JNK/p38 MAPK和PI3K/Akt訊息路徑可能在大腸癌細胞的增生與遷移扮演著重要的角色。此外，肉桂醛能夠明顯地削弱
細胞的增生與遷移有可能是透過抑制這兩種訊息傳導路徑以及細胞週期進行、甚至促進細胞凋亡。
Colon cancer is a cancer from uncontrolled cell growth in the colon and rectum. In recent years, colon cancer is the second leading cause of cancer mortality in the United States. According to the increase in elder population and fat intake and the decline in intake of fiber and eating habits westernized, thus, not only the increase in cardiovascular disease but also the number of patients with colon cancer has continued to increase in Taiwan. Cinnamaldehyde is an organic compound, yellow viscous liquid, which is the major and bioactive compound isolated from the leaves of Cinnamomum osmophloeum kaneh. Several studies have demonstrated that cinnamaldehyde has anti-bacterial effects, immunomodulatory, anti-fungal activities, and anti-diabetic nephropathy effects. The consequences of in vitro treatment with cinnamaldehyde in different cells or tissues have diverse responses and these mechanisms are very complicated.
In the present study, the role of cinnamaldehyde in proliferation and migration in human colon cancer cells was examined. From the results of MTT assay and cellular number analysis, we found that cinnamaldehyde (100 μM) treatment significantly decreased cellular growth compared with normal culture condition when the incubation period was for 72 h in HT-29 and SW-480 human colon cancer cells. The reduced rate of cell survival was about 30%. No significant changes in cell viability when the incubation period was for 24 or 48 h. Moreover, inhibition of cell migration was displayed in cinnamaldehyde (100 μM), the ERK inhibitor PD98059 (5 μM), the JNK inhibitor SP600125 (2 μM), the p38 MAPK inhibitor SB203980 (2 μM), the PI3K inhibitor LY294002 (5 μM), and the Akt inhibitor SH-5 (2 μM) treatments in these cells. Interestingly, activation of the ERK/JNK/p38 MAPK and the PI3K/Akt signaling pathways were attenuated by cinnamaldehyde. We suggested that cinnamaldehyde suppressed cell proliferation and migration through these signaling pathways in human colon cancer cells. On the other hand, cinnamaldehyde caused inhibition of cellular mitogenesis partly by promoting apoptosis. Amplified changes in caspase 3 activity, Bcl-2 protein expression, PARP cleavage, DNA fragmentation, and mitochondrial cytochrome c release were showed in cinnamaldehyde treatments in these cells. The ability of cinnamaldehyde to induce cell cycle arrest was also verified by the observation that it significantly decreased the protein level of PCNA but increased the protein levels of p27Kip1 and p21Waf1/Cip1.
It is concluded that both the ERK/JNK/p38 MAPK and the PI3K/Akt signaling pathways were examined to play important roles in cell proliferation and migration in human colon cancer cells. Cinnamaldehyde significantly attenuated cell proliferation and migration at least partly by inhibiting these signaling pathways and cell cycle progression, and promoting apoptosis